Triple negative breast cancer initiating cell subsets differ in functional and molecular characteristics and in γ-secretase inhibitor drug responses

Increasing evidence suggests that stem-like cells mediate cancer therapy resistance and metastasis. Breast tumour-initiating stem cells (T-ISC) are known to be enriched in CD44⁺CD24^neg/low cells. Here, we identify two T-ISC subsets within this population in triple negative breast cancer (TNBC) lines and dissociated primary breast cancer cultures: CD44⁺CD24^low+ subpopulation generates CD44⁺CD24^neg progeny with reduced sphere formation and tumourigenicity. CD44⁺CD24^low+ populations contain subsets of ALDH1⁺ and ESA⁺ cells, yield more frequent spheres and/or T-ISC in limiting dilution assays, preferentially express metastatic gene signatures and show greater motility, invasion and, in the MDA-MB-231 model, metastatic potential. CD44⁺CD24^low+ but not CD44⁺CD24^neg express activated Notch1 intracellular domain (N1-ICD) and Notch target genes. We show N1-ICD transactivates SOX2 to increase sphere formation, ALDH1+ and CD44⁺CD24^low+cells. Gamma secretase inhibitors (GSI) reduced sphere formation and xenograft growth from CD44⁺CD24^low+ cells, but CD44⁺CD24^neg were resistant. While GSI hold promise for targeting T-ISC, stem cell heterogeneity as observed herein, could limit GSI efficacy. These data suggest a breast T-ISC hierarchy in which distinct pathways drive developmentally related subpopulations with different anti-cancer drug responsiveness.